This invention relates to nucleic acid molecules and amino acid sequences of a new mammalian protein and to their use in the characterization, diagnosis, and treatment of conditions such as Alzheimer""s disease, Down syndrome and other forms of dementia.
Alzheimer""s disease is a progressive neurodegenerative disorder that is characterized by the formation of senile plaques and neurofibrillary tangles containing amyloid beta peptide. These plaques are found in limbic and association cortices of the brain, including hippocampus, temporal cortices, cingulate cortex, amygdala, nucleus basalis and locus caeruleus. Early in Alzheimer""s pathology, physiological changes are visible in the cingulate cortex (Minoshima et al. (1997) Annals of Neurology 42:85-94). In subjects with advanced Alzheimer""s disease, accumulating plaques damage the neuronal architecture in limbic areas and eventually cripple the memory process.
Approximately twenty million people worldwide suffer with dementia that results from Alzheimer""s disease. The disease can be early onset affecting individuals as young as 30 years of age, or it can be familial or sporadic. Familial Alzheimer""s disease was once thought to be inherited strictly as an autosomal dominant trait; however, this view is changing as more genetic determinants are isolated. For example, some normal allelic variants of apolipoprotein E (ApoE), which is found in senile plaques, can either protect against or increase the risk of developing the disease (Strittmatter et al. (1993) Proc Natl Acad Sci 90:1977-1981).
Mutations in four genes are known to predispose an individual to Alzheimer""s disease: ApoE, amyloid precursor protein (APP), presenilin-1, and presenilin-2 (Selkoe (1999) Nature 399:A23-A31). The e4 allele of the ApoE gene confers increased risk for late onset Alzheimer""s disease. xcex2-amyloid protein (Axcex2) is the major component of senile plaques, and it is normally formed when xcex2- and xcex3-secretases cleave APP. In Alzheimer""s disease patients, large quantities of Axcex2 are generated and accumulate extracelluarly in these neuropathological plaques.
Associations between Alzheimer""s disease and many other genes and proteins have been reported. Fetal Alzheimer antigen an synuclein a are found in brain plaques and tangles. Inheritance of some gene polymorphisms is also linked to increased risk of developing the disease. For example, a polymorphism in the gene encoding xcex22-macroglobulin, a protein that can act as a protease inhibitor, is associated with increased risk for developing a late-onset form of Alzheimer""s disease.
Experiments using microarray technology have provided additional evidence for changes in expression of specific genes in tissues from subjects with Alzheimer""s disease. One such gene, Down syndrome critical region 1-like 1 (Mazowiecki et al. (1996) J Biol Chem 271:14567-14571) also named DSCR1L1 (g1435040), was found to be down-regulated more than two-fold in Alzheimer""s tissue. DSCR1L1 encodes a thyroid hormone responsive protein and is a member of a gene family that includes DSCR1 (g7657042) and DSCR1L2 (g6017918). The first member of the family cloned, DSCR1, was named based on its proximity to the Down syndrome region of chromosome 21, but the function of the encoded protein and its role in Down syndrome, if any, remains unclear. The defining motifs of this family of proteins include an N-terminal RNA-binding domain, which is similar to those found in many RNA-binding proteins and in some single-stranded DNA-binding proteins, and a central short, unique serine-proline motif that includes an ISPPXSPP box that may be a target for phosphorylation (Fuentes et al. (1995) Hum Mol Genet 4:1935-1944).
Based on general features of the amino acid sequences, these DSCR1 genes likely encode proteins involved in transcriptional regulation and signal transduction (Strippoli et al. (2000) Genomics 64:252-263). The three genes appear to be differentially expressed: DSCR1 is highly expressed in fetal brain and heart, DSCR1L1 is evident in heart, brain, liver and skeletal muscle, and DSCR1L2 is expressed in most tissues including blood.
There are several connections between Alzheimer""s disease and Down syndrome, such as the appearance of enhanced xcex2-amyloid deposits in middle-aged Down syndrome patients. The thyroid hormone responsive properties of DSCR1L1 may be relevant to the role for this gene in Alzheimer""s disease and/or Down syndrome: both Alzheimer""s and Down""s patients have been shown to have disturbances in thyroid hormone metabolism including elevated antithyroglobulin antibodies and reduced levels of prealbumin in the cerebrospinal fluid of Alzheimer""s patients and pronounced subclinical hypothyroidism in Down""s patients who have clinical manifestations of Alzheimer""s disease (Sutherland et al. (1992) Neurobiol Aging 13:301-312). Conversely, hypothyroid patients display some of the same neurologic symptoms seen in Alzheimer""s patients and a history of thyroid dysfunction is considered to be a risk factor for developing Alzheimer""s disease. One possible connection is the discovery that thyroid hormone negatively regulates the transcriptional activity of the APP gene (Belandia et al. (1998) J Biol Chem 273:30366-30371); overexpression of APP has been hypothesized to cause neuronal degeneration by a mechanism involving increased production of xcex2-amyloid protein. The down-regulation of the thyroid responsive DSCR1L1 gene and possibly other DSCR1 genes in Alzheimer""s patients also provides a link between Alzheimer""s and thyroid hormone function.
The discovery of additional mammalian nucleic acid molecules encoding members of the DSCR1 protein family may be used for the diagnosis, prognosis or treatment of Alzheimer""s disease, Down syndrome and other forms of dementia.
The invention is based on the discovery of a substantially purified mammalian nucleic acid molecule encoding mammalian DSCR1L1xcex1 protein, which satisfies a need in the art by providing compositions useful in the characterization, diagnosis, and treatment of conditions such as Alzheimer""s disease, Down syndrome and other forms of dementia.
The invention provides an isolated mammalian cDNA or a fragment thereof encoding a mammalian protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:2, a variant having at least 95% identity to the amino acid sequence of SEQ ID NO:2, and an antigenic epitope of SEQ ID NO:2. The invention also provides an isolated mammalian cDNA or the complement thereof selected from the group consisting of a nucleic acid sequence of SEQ ID NO:1, a variant having at least 85% identity to the nucleic acid sequence of SEQ ID NO:1, and a fragment of SEQ ID NO:1. The invention additionally provides a composition, a substrate, and a probe comprising the cDNA, or the complement of the cDNA, encoding DSCR1L1xcex1. The invention further provides a vector containing the cDNA, a host cell containing the vector and a method for using the cDNA to make DSCR1L1xcex1. The invention still further provides a transgenic cell line or organism comprising the vector containing the cDNA encoding DSCR1L1xcex1. The invention additionally provides a mammalian fragment or the complement thereof selected from the group consisting of SEQ ID NOs:3-9. In one aspect, the invention provides a substrate containing at least one of these fragments. In a second aspect, the invention provides a probe comprising the fragment which can be used in methods of detection, screening, and purification. In a further aspect, the probe is a single stranded complementary RNA or DNA molecule.
The invention provides a method for using a cDNA to detect the differential expression of a nucleic acid in a sample comprising hybridizing a probe to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with a standard, wherein the comparison indicates the differential expression of the cDNA in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing differential expression of the cDNA is used to diagnose Alzheimer""s disease, Down syndrome and other forms of dementia. In another aspect, the cDNA or a fragment or a complement thereof may comprise an element on an array.
The invention additionally provides a method for using a cDNA or a fragment or a complement thereof to screen a library or plurality of molecules or compounds to identify at least one ligand which specifically binds the cDNA, the method comprising combining the cDNA with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the cDNA, thereby identifying a ligand which specifically binds the cDNA. In one aspect, the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, transcription factors, repressions, and regulatory molecules.
The invention provides a purified mammalian protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:2, a variant having 95% identity to the amino acid sequence of SEQ ID NO:2, an antigenic epitope of SEQ ID NO:2, an oligopeptide of SEQ ID NO:2, and a biologically active portion of SEQ ID NO:2. The invention also provides a composition comprising the purified protein or a portion thereof in conjunction with a pharmaceutical carrier. The invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. In one aspect, the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs. In another aspect, the ligand is used to treat a subject with Alzheimer""s disease, Down syndrome or other forms of dementia.
The invention provides a method of using a mammalian protein to screen a subject sample for antibodies which specifically bind the protein comprising isolating antibodies from the subject sample, contacting the isolated antibodies with the protein under conditions that allow specific binding, dissociating the antibody from the bound-protein, and comparing the quantity of antibody with known standards, wherein the presence or quantity of antibody is diagnostic of Alzheimer""s disease, Down syndrome and other forms of dementia. The invention also provides a method of using a mammalian protein to prepare and purify antibodies comprising immunizing a animal with the protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified antibodies.
The invention provides a purified antibody which bind specifically to DSCR1L1xcex1. The invention also provides a method of using an antibody to diagnose Alzheimer""s disease, Down syndrome and other forms of dementia comprising combining the antibody comparing the quantity of bound antibody to known standards, thereby establishing the presence of the disease. The invention further provides a method of using an antibody to treat Alzheimer""s disease, Down syndrome and other forms of dementia comprising administering to a patient in need of such treatment a pharmaceutical composition comprising the purified antibody.
The invention provides a method for inserting a marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide. The invention also provides a method for using a cDNA to produce a mammalian model system, the method comprising constructing a vector containing the cDNA selected from SEQ ID NOs:1, and 3-9, transfonning the vector into an embryonic stem cell, selecting a transformed embryonic stem, microinjecting the transformed embryonic stem cell into a mammalian blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, mammalian model system.